Hepatitis C virus (HCV) infects approximately 2% of the world's population and is the primary cause of liver transplants in the United States. Based on lessons learned from diseases such as AIDS, HCV RNA replication is a promising target for antiviral development. However, the replication of all viruses with plus-strand RNA genomes is poorly understood, especially at the biochemical level. The overall goal of the research in the Kao lab is to understand mechanism of RNA virus replication. The goal for this project is to build knowledge about the subunits of the HCV replication complex, with emphasis on protein-RNA, and protein-protein interactions. This is an extension of the past six years of research in the Kao lab, where a number of basic properties of the HCV polymerase and the HCV protease-helicase have been examined using biochemical, biophysical, and cell-based methods. The research can be partitioned into several related subaims that will: 1. Identify and validate the biological importance of the residues in the HCV RdRp that interacts with the substrate NTPs, the template RNA, and during different stages of HCV RNA synthesis. 2. Elucidate the interactions between the HCV RdRp and the nascent RNA and identify and characterize the nascent RNA exit channel. 3. Examine the protein-protein and protein-RNA interactions with other replicase- associated subunits of the HCV replicase and examine effects of the interactions on HCV replicase formation in cells. 4. Obtain images of the protein complexes using electron microscopy and reconstruct their structures. Results from this proposal will advance the understanding of the mechanism of viral RNA-dependent RNA synthesis for ALL positive-strand RNA viruses. The knowledge can also be used to compare the mechanisms of action of all template-dependent (both viral and cellular) polymerases.